Bacterial spore envelope

C. Rodrigues, Warwick University, UK
Benoit Gallet & Christine Moriscot (Team headed by G. Schoehn, IBS)

Bacterial spores are dormant cells that can withstand a wide range of stresses, including antibiotics, detergents, irradiation and high temperatures. Such resilience is an asset when spores are used for the benefit of humans (e.g. probiotics, spore-based delivery technologies) but it represents a major problem in terms of infectious diseases, food safety or biological warfare when it comes to spores of pathogenic bacteria (e.g. spores of Bacillus anthracis or Clostridium difficile).

Upon environmental stress, spore-formers enter a differentiation process, starting with an asymmetric division (stage I) that results in two morphologically different compartments : a large mother cell and a small forespore, into which a full copy of the chromosome is translocated (stage IIE). These two cells are genetically identical but they follow specific gene expression programs, governed by tightly regulated sporulation-specific sigma factors (σ).
Following asymmetric division, the mother cell and forespore are separated by the so-called intermembrane space (IMS), composed of two membranes and peptidoglycan (PG). The two cells then undergo dramatic morphological changes as the forespore is progressively internalized into the mother cell through a phagocytic process called engulfment (stage IIM), which requires coordinated PG synthesis and degradation. The engulfed forespore is eventually surrounded by its own cytoplasmic membrane and a second membrane derived from the mother cell (stage III). Tethering of these two membranes involves the assembly of a transenvelope nanomachine called the SpoIIIA-SpoIIQ complex (A-Q complex), which is also required to maintain the forespore physiology. Halfway through engulfment, protective protein layers (called the coat) begin to self-assemble at the surface of the forespore. At the end of engulfment, a modified PG, called the cortex, is synthesized in the IMS. The mature spore is eventually released into the environment upon mother cell lysis (stage VI). The spore can remain dormant for thousands of years, while still being receptive to its environment, so that it can germinate and resume vegetative growth under appropriate conditions.

Despite their importance for the acquisition of resistance properties, the mechanisms involved in spore development are not yet fully elucidated, mainly because they involve macromolecular complexes of nanometric dimensions, whose assembly usually requires the cellular environment.
A first example is the transmembrane multi-protein A-Q complex (> 2 MDa). In its absence, the spore exhibits shape defects and fails to acquire the ability to withstand extreme environments. The structure and function of the A-Q complex remain enigmatic, but its structural similarities with specialized secretion systems and proton pumps suggest that it could be a new type of transport machinery, enabling the mother cell to "feed" the spore or transport specific molecules between the two cellular compartments (Morlot and Rodrigues, Trends Microbiol, 2018). We study this complex in Bacillus subtilis to unravel its structural and evolutionary relationship to other transport systems, and to determine the nature of the secreted molecule and the role it plays during spore development (Collab. C. Rodrigues, Warwick Univ.). We have so far discovered that the A-Q component called SpoIIIAG (AG) forms large homo-oligomeric rings whose architecture and dimensions are reminiscent of ring-forming components found in type-III secretion systems (Rodrigues et al., PNAS, 2016). In addition, our structural characterization of various proteins associated with the A-Q complex has provided insights into the oligomerization ability of ring-building motifs found in various transport machineries, and opened up avenues of research into alternative functions (Trouve et al., J. Struct. Biol., 2018 ; Liu et al., J. Struct. Biol., 2022).
We are now developing cryo-tomography approaches to study the structure of the A-Q complex in sporulating cells.

Liu et al., J. Struct. Biol., 2022
Trouve et al., J. Struct. Biol., 2018
Rodrigues et al., PNAS, 2016

Among the determinants of spore resistance is the extracellular shell made of proteinaceous layers called the coat. Its assembly is based on a complex network of interactions involving first a tens morphogenetic proteins, and eventually more than 80 different proteins. Despite their importance for the acquisition of resistance properties, the architecture of the different coat layers remains poorly understood, because their deposition and maturation is a long (> 7 hours) and complex process. In collaboration with the group of Guy Schoehn at the IBS, we use cryo-electron tomography (cryo-ET) on spore lamellae generated by cryo-FIBM/SEM (cryo-focused ion beam milling coupled to scanning electron microscopy) to investigate coat formation in B. subtilis. We have recently shown that in early stages of sporulation, the nascent coat is made of a stack of distinct embryonic layers, whose architecture requires specific morphogenetic proteins.
This work lays the foundations for the dissection of molecular mechanisms involved in the development and the resistance of the bacterial spore. Our next challenging objective is to develop super-resolution cryo-CLEM (cryo-PALM coupled to cryo-FIB/SEM and cryo-tomography) to unravel the structure and composition of the coat layers all along the sporulation cycle.

Coupe à travers un tomogramme (gauche) d’une cellule sporulante de B. subtilis utilisée pour la segmentation (centre et droite) de diverses ultrastructures de la pré-spore et cellule mère.

Bauda et al. Nat. Commun., 2024.